Pathomechanistic Networks of Motor System Injury in Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is the most common, adult-onset, progressive motor neurodegenerative disorder that results in death within 3 years of the clinical diagnosis. Due to the clinicopathological heterogeneity, any reliable biomarkers for diagnosis or prognosis of ALS have not been identified till date. Moreover, the only three clinically approved treatments are not uniformly effective in slowing the disease progression. Over the last 15 years, there has been a rapid advancement in research on the complex pathomechanistic landscape of ALS that has opened up new avenues for successful clinical translation of targeted therapeutics. Multiple studies suggest that the age-dependent interaction of risk-associated genes with environmental factors and endogenous modifiers is critical to the multi-step process of ALS pathogenesis. In this review, we provide an updated discussion on the dysregulated cross-talk between intracellular homeostasis processes, the unique molecular networks across selectively vulnerable cell types, and the multisystemic nature of ALS pathomechanisms. Importantly, this work highlights the alteration in epigenetic and epitranscriptomic landscape due to gene-environment interactions, which have been largely overlooked in the context of ALS pathology. Finally, we suggest that precision medicine research in ALS will be largely benefitted from the stratification of patient groups based on the clinical phenotype, onset and progression, genome, exposome, and metabolic identities.


INTRODUCTION
Amyotrophic Lateral Sclerosis (ALS) is the third most common type of neurodegenerative disorder and the most common type of 'Motor Neurone Disease' (MND) [1].The global incidence of ALS currently ranges between 0.6 and 3.8 per 100,000 person-years, which is increasing over time [2].The prevalence of ALS is approximately 4.1-8.4 per 100,00 persons, with a lifetime risk of 1 in 350 and a median onset age of 51-66 years [3].ALS initiates focally and asymmetrically in the upper and/or lower limbs or in the bulbar regions, followed by a gradual progression to the contiguous voluntary muscles [4].Moreover, about 28% of sporadic and 48% of familial cases of ALS display cognitive deficits associated with frontotemporal dementia (FTD) [5].Clinical hallmarks of 'classical' ALS cases involve progressive and concomitant degeneration of upper (UMN) and *Address correspondence to this author at the Department of NMR Microimaging and Spectroscopy, CSIR-Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad 500007, Telangana, India; E-mail: abpatel@ccmb.res.inlower motor neurons (LMN) in the motor cortex, brain stem, and spinal cord [4].Early clinical signs of UMN and/or LMN defects are followed by a progressive paralysis that causes death by respiratory failure in ≥ 50% of patients within 2-5 years of diagnosis [6].Major sub-clinical signatures in ALS involve atrophy of the primary motor cortex and ventral projections of the spinal cord, degeneration of the corticospinal and corticobulbar tracts, degeneration of the hypoglossal-trigeminal-phrenic nerves of the brain stem, and wasting of the somatic and bulbar skeletal muscles [7].Underlying microstructural abnormalities include massive death of spinal -motor neurons and varying loss of corticomotor Betz cells, with diffuse gliosis in the subcortical white matter and the grey matter of the motor cortex and spinal cord [7].
Gender-based risk of ALS incidence is approximately double for the male sex [8], with a higher mean age at diagnosis and a lower peak age of prevalence among women [9].Interestingly, a lower survival time with a higher risk of bulbar onset has been reported in women [10].However, these gender ratios might change with the age of incidence, both geographically and temporally [11].A higher incidence in Europe compared to South and East Asia has been correlated with the lower prevalence of known ALS genes in Asian populations [3,11].The presence of a prodromal delay, respiratory or genitourinary comorbidities, gradual weight loss or poor nutritional status, and cognitive impairment or depressive disorders tend to negatively affect survival in ALS patients [3].Based upon their representativeness across various population-based studies, the above factors can act as the demographic variants that skew the association of disease with prognostic factors.
Due to a wide variety of 'ALS-mimic' disorders, the use of a range of diagnostic methods involving clinical, electrophysiological, neuroimaging, and neuropathological tests is required to ascertain the ALS 'syndrome' [12].A definitive diagnosis of ALS requires the revised El Escorial criteria with the revised Airlie House criteria from the World Federation of Neurology (WFN), the Awaji-Shima criteria, and the Gold Coast criteria [13][14][15].
As revealed by various large-scale epidemiological studies, a wide range of susceptibility factors and resistance mechanisms contribute to the variability of disease progression and survivability in ALS [4].The complex etiology of the ALS spectrum is believed to be the result of a timevariable cross-talk between environmental risk factors and endogenous metabolic modifiers, with diverse types of genetic polymorphisms having different cytotoxic or cytoprotective effects depending on neuronal types [16].
In this review, we highlight the complex interplay of impaired cellular processes that define the pathomechanistic signature during ALS onset and progression.This majorly involves synaptic failure, neuroinflammation, dysregulated proteostasis and RNA metabolism, impaired endosomal trafficking and axonal transport, oxidative stress, DNA damage, and metabolic dyshomeostasis [17].The complex genetic architecture of ALS dictates the molecular heterogeneity of affected cellular networks and generates a 'pathological continuum' of clinical phenotypes.Moreover, this review draws a focus on the multisystem nature of pathological processes, the selective vulnerability of motor neuron and myofibre subtypes, and the dysregulated neuroglial and neuromuscular networks in ALS 'syndrome' [18].
Additionally, hurdles in unraveling the 'missing heritability' of ALS pathogenesis can be overcome by looking in the direction of DNA or RNA modifications associated with the disease.Here, we describe the critical role of geneenvironment interaction in disease precipitation in conjunction with the dysregulated epigenetic machinery and epitranscriptomic landscape in ALS.Finally, we suggest that the need of the hour is to discern how the pathological cellular networks work simultaneously, sequentially, or synergistically in ALS.

GENETIC RISK FACTORS OF ALS
Traditionally, over 90-95% of cases of ALS are determined to be sporadic (sALS), and only 5-10% of cases have a familial origin (fALS), usually with an autosomal dominant inheritance pattern [19].Over the years, >50 potentially causative or disease-modifying genes associated with ALS have been identified [20].The genetic component in 60-70% of fALS cases is known, while the heritability of sALS cases lies at 50% [21].Approximately 50% of fALS cases and 5% of sALS cases are attributed to high-risk genes such as chromosome 9 open reading frame 72 (C9ORF72), superoxide dismutase (SOD1), TAR-DNA binding protein (TARDBP) and fused in sarcoma (FUS) [20].
Many of the fALS-associated mutations have now been reported in sALS cases, being previously undetected due to incomplete genotypic penetrance, delayed disease onset, and/or oligogenic contribution [23].Analyses of various large-scale genome-wide association studies contribute to the 'multiple rare variants, common disease' hypothesis of sporadic ALS [24].Moreover, the genetic contribution to the risk of ALS onset and the rate of progression seems to be independent of each other [25].It is yet unclear how the mutations in individual ALS-associated genes having divergent cellular functions ultimately converge on the signature disease phenotype.It has become apparent that systematic genetic profiling of both familial and sporadic ALS patients followed by a genetic sub-classification is crucial for the development of targeted therapeutics and relevant preclinical models for drug screening.

PATHOPHYSIOLOGY OF ALS
Contiguity of the degenerative spreading through the central nervous system (CNS) is indicated by the decline in the integrity of multiple interconnected micro-circuits within and associated with the motor network [26].These circuits involve 'determinants' such as the quadripartite network of neuron-oligodendrocyte-astrocyte-microglia cell types, the neuromuscular synapse, the inhibitory feedback connections with interneurons, as well as 'contributors' across the neurovascular units such as lymphocytes and pericytes [26].Here, we discuss the currently available knowledge of the pathophysiological mechanisms of ALS.

Multifaceted Pathology of Synaptic Failure
ALS is often regarded as a synaptopathy, wherein motor degeneration is preceded by a loss of synaptic scaling and plasticity [26,27].This occurs with concomitant regression and degeneration of synaptic architecture and apical dendrites [27,28].
Altogether, the gain of excitatory drive and loss of inhibitory regulation increases Ca 2+ influx and triggers a sustained neuronal depolarization [35].Due to the lack of Ca 2+buffering proteins, defects in the Ca 2+ ATPase pump and Ca 2+ /Na + exchangers, and upregulation of voltage-gated Ca 2+ channels, the neuronal hyperexcitability disrupts the intracellular Ca 2+ homeostasis cycle across the endoplasmic reticulum (ER) and mitochondria [36,37].This leads to cellular ATP depletion and increased ROS production, which in turn triggers Na + /K + ATPase pump dysfunction and further enhances neuronal firing [28].Thus, the local motor circuitry is compromised to glutamate excitotoxicity.Notably, there are differences in opinion regarding the region of origin and the course of disease progression in ALS, influenced by the variable vulnerability of motor neurons and muscle fibre types (Fig. 1).

Effects on Neuromuscular Junctions
Mutant protein aggregates of SOD1 in skeletal muscles sequester cytoskeletal proteins like kinesin-associated protein (KAP3) [38].Along with the downregulation of the acetylcholine transporter ChAT, this process inhibits acetylcholine (ACh) release from LMNs [38].Overall, it results in the dismantling of neuromuscular junctions (NMJ) and a loss of myofibre contractility [39].

Selective Vulnerability of Motor Neuron and Skeletal Myofibre Subtypes
ALS-vulnerable fast-firing fast-fatigable (FF) motor neurons have larger cell bodies, faster axonal conduction velocity, elaborate dendritic branching, and high NMJ innervation as compared to the ALS-resistant slow-twitch fatigueresistant (S-type) motor neurons [51].FF motor neurons (MNs) innervate the fast-contracting extrafusal myofibres, while S-type neurons connect with the slow intrafusal fibres [37].Moreover, ALS-vulnerable MNs are susceptible to oxidative stress and proteostasis dysregulation due to the low levels of free-radical scavengers and cytosolic calcium buffering proteins (e.g., parvalbumin, calbindin, calreticulin) [52].
Overactivation of osteopontin-integrin-matrix metalloprotein 9 axis is involved in ER-stress-induced inflammation in FF neurons, with a concomitant upregulation of neurofilament proteins of retrograde transport.Moreover, upregulation of SIL1, the co-chaperone of ER-resident binding immunoglobulin protein (BiP), induces unfolded protein response (UPR); while the insulin growth factor (IGF-2/IGF-1) signalling protects against glutamate excitotoxicity in S-type neurons [37].FF neurons express the Na + /K + ATPase-α3 subunit that is selectively impaired by mutant proteins, while S-type neurons express the resilient Na + /K + ATPase-α1 subunit [53].Neuronal vulnerability is also dictated by the differential expression of GABA A -α2 and GLUR1 on FF neurons, with GABA A -α1 and GLUR2 on S-type neurons [54].Notably, the apoptosis-inducing factor SEMA3A is secreted by the ALS-vulnerable type-IIb fast-twitch glycolytic myofibres, while the ALS-resistant type-I slow-twitch oxidative myofibres secrete SEMA3E [51].Intriguingly, presynaptic Na + channels (ENaC) can slow down the synaptic dysfunction by inducing a non-selective 'presynaptic homeostatic plasticity' at ALS-affected NMJs [55].
Although the dysfunction of motor circuitry is profound in ALS, there is evidence of sensory neuronal abnormalities across the heterogenous population of ALS patients.This enforces the idea of the multisystem nature of disease progression in ALS (details in Box 1).

Fig. (1).
Pattern of the motor system dysfunction in ALS.Progressive degeneration affects the vulnerable fast-fatigable (FF) motor neurons and type IIb myofibres.Slow twitch (S) motor neurons and type I myofibres are resilient until the end stages.A 'dying forward' hypothesis proposes an anterograde degenerative trigger from cortex progresses towards spinal cord through trans-synaptic glutamate excitotoxicity.LMNs without a monosynaptic connection to corticomotor neurons are resilient to ALS pathology such as oculomotor, abducens, trochlear nerve, and Onuf's nuclei.A 'dying back' hypothesis suggests that an early retrograde degeneration trigger from neuromuscular junctions .(NMJs) moves back towards primary motor cortex by distal axonopathy Both UMN and LMN degeneration can also occur independently Reprinted from N Engl and in a stochastic manner during ALS progression, which might be attributed to oxidative stress mediated apoptosis.J Med 377:2, Brown, R.H.; Al-Chalabi, A., Amyotrophic Lateral Sclerosis, 162-172, Copyright (2017), with permission from (Massachusetts Medical Society).(A higher resolution/colour version of this figure is available in the electronic copy of the article).

Box 1. Sensory Dysfunction in ALS
Dysfunction in groups of neurons within extramotor areas such as the frontotemporal cortex, substantia nigra, thalamus, hippocampal dentate granule layers, spinocerebellar pathways, autonomic system, dorsal columns, and peripheral sensory nerves has been observed across ALS patient subgroups [56].
• Although true sensory neuropathy is rare in ALS, subjective and objective sensory symptoms occur in ~50% and ~10% of patients, respectively [57].Presymptomatic impairment is reported in spindle-afferent proprioceptive sensory neurons of dorsal root ganglia in SOD1 G93A mice [58].
• Mutant proteins accumulate in the soma of sensory neurons in presymptomatic SOD1 G93A mice, whereas TDP43 A315T mutant protein dysregulates ER-stress response and stress granule assembly in sensory neurons [59].Mutant TDP43 causes axonal accumulation of RNA binding proteins, which triggers distal axonopathy of sensory neurons [59].Along with local and long-range alterations in sensorimotor circuits, these findings enforces the multisystem nature of ALS [57].
At presymptomatic stage, the synaptic dysfunction of fast fatigable α-MNs reduces the activity of fast contractile myofibres, and induces an increase in glutamatergic spindleafferent inputs to α-MNs.This process triggers the excitotoxic death [60].Hence, it is not clear whether hyperexcitability triggers neurodegeneration, or it arises to compensate neuronal death [61].
It is important to note that the differences in vulnerability of specific motor neuron or muscle fibre types potentially regulate the dominant heterogeneity in UMN and LMN involvement across various ALS phenotypes depending on the site of onset.A new line of ALS research is currently focused on targeting the unique structural, functional, and molecular features of these cell types for developing targeted preventive or therapeutic measures.

Non-cell Autonomous Neuroinflammation
Reactive microgliosis progresses in motor and even extra motor areas since the early presymptomatic stage [62].It is preceded by reactive astrocytosis with regionally variable activation through inherent defects or paracrine signalling from neurons [63].Given the dual role of astroglia in neuroprotection and neurotoxicity, a chronic disease state can drive an adaptive CNS-specific response towards systemic hyper-inflammation [64].

Role of Blood-CNS Barrier in Neuroinflammation
In medulla oblongata and spinal cord from sALS patients and transgenic SOD1 mice, the presence of dysfunctional microvessels indicates pervasive damage to the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) with micro-haemorrhages [81].This phenomenon hampers the recruitment of anti-inflammatory macrophages and Treg cells and induces a toxic neurovascular inflammatory response [81].Ultrastructural analysis of the choroid plexus in ALS patients revealed the loss of tight junction components, disruption of vascular integrity, vascular injury, and loss of pericytes [82].
Currently, the initiation of immune response and the nature of the involvement of specific immune cells in ALS onset and progression have not been completely understood.This is because of the chronic nature and late diagnosis of ALS, which makes it difficult to determine whether abnormal immune response or inflammation is a cause or consequence of the disease [83].

Role in Loss of Neuroprotection
In ALS, there is a disruption of the metabolic coupling of spinal neurons with the astrocytes and oligodendrocytes.This results from the decreased transport of glycolytic products (lactate) for neuronal oxidative phosphorylation due to the loss of the astrocytic (MCT4) and oligodendrocytic (MCT1) lactate efflux transporters [90].Together with the pro-nerve growth factor (pro-NGF) and p75 neurotrophin receptor (p75 NTR ) signalling in astrocytes during mitochondrial dysfunction, the neuroglial metabolic uncoupling triggers microglial activation [90].There is a reduced secretion of astrocytic trophic factors that mediate neuroprotective cross-talk between astroglia and neurons such as BDNF, VEGF, NGF, plasminogen, basic fibroblast growth factor (bFGF), and neurotrophin 3/4 (NT-3/4) [91].Death of oligodendrocytes reduces the secretion of trophic factors such as GDNF and ciliary neurotrophic factor (CNTF) to promote neurodegeneration [92] Interestingly, OPTN is known to inhibit NF-κB mediated astroglial activation, glutamate excitotoxicity, chronic inflammation, and necroptosis.Targeted depletion of OPTN in oligodendrocytes and microglia leads to reactive gliosis with demyelination and axonopathy [93].Notably, microglial activation is facilitated by the necroptotic death of neurons and oligodendrocytes at early stages (details in Box 2).

Box 2. Necroptosis: A Road to ALS Neuroinflammation
Motor neurons: At early stages, intrinsic genetic defects and environmental insults can trigger the necroptosis of a small number of motor neurons (MNs).This results in the autophagymediated release of MN-specific antigens and pro-inflammatory stimuli (e.g., ATP, alarmins, and damaged mitochondria) [94].MN-antigen-specific T-helper (Th17) cells are recruited into the central nervous system so that their interaction with astroglial cells initiates an adaptive immune response to target the neighbouring MNs [78].This is facilitated by a concurrent infiltration of innate immune cells such as macrophages, natural killer cells, neutrophils, and mast cells [95,96].Therefore, necroptosis plays a major role during disease initiation, whereas disease progression is majorly mediated by immune dysfunction [93].
Oligodendrocytes: Necroptosis of oligodendrocytes is triggered by mutant protein aggregates and reactive microglial signalling prior to neurodegeneration [67,97].Reactive astrocytes hinder axonal remyelination via a dysfunctional differentiation of oligodendrocyte precursor cells (OPCs) [98].In a conditional knock-out mouse model of OPTN, the loss of TBK1-regulated type-I interferon response and necroptotic signalling results in the death of spinal oligodendrocytes [93].
Deletion of mutant SOD1 allele from motor neurons and oligodendrocytes in a transgenic mouse model of ALS delays disease onset but not progression [102,103].Mutant SOD1 depletion from microglia slows down disease progression by 50% but not the onset [104].Targeted overexpression of mutant SOD1 in astrocytes and oligodendrocytes does not trigger the ALS phenotype.This highlights the need for distinct therapeutic interventions targeting the non-cell autonomous pathology at definite stages of the disease [105].

Dysregulation of Autophagy
Motor neurons (MNs) are terminally differentiated cells that heavily depend on constitutive autophagy for the clearance of misfolded protein aggregates, accumulated stress granules, damaged organelles, and defective cargo-loaded presynaptic vesicles [106].Interestingly, autophagy displays a dual mode of action in ALS pathology.Activation of au-tophagy protects MNs and NMJs from an accumulation of mutant protein aggregates at the presymptomatic stage, whereas it mediates disease progression at the symptomatic stage.In spinal neurons and senescent skeletal muscles of ALS patients and mouse models, the accumulation of autophagosomes in the inclusion bodies at the symptomatic stage indicates an impairment of autophagy [107,108].Furthermore, transcription-independent accumulation of earlystep autophagy factors (e.g., Beclin-1, p62/SQSTM1, LC3-II, Atg5-Atg12 complex) is observed in motor neurons at the presymptomatic stage and in astroglia at later stages [109].
Over-induction of autophagy occurs as an adaptive response to a concomitant milieu of cellular stressors such as DNA damage stress, proteotoxic stress, ER stress, and oxidative stress.Increased autophagosome formation exceeds the lysosomal degradation rate and subsequently triggers apoptosis and type II cell death (autophagic-lysosomal) in various neurodegenerative disorders [110].This process is associated with the downregulation of BDNF and its receptor tropomyosin receptor kinase B (TrkB) signalling, which otherwise protects neurons through the phosphatidylinositol-3-kinase/ protein kinase B/mammalian target of rapamycin (PI3K/ PKB/mTOR) pathway [111].Age-associated impairment of lysosomal activity occurs due to the intra-lysosomal aggregates of lipofuscin granules, various extra-lysosomal aggregates, and damaged mitochondria.Altogether, these processes inhibit autophagy, which potentially contributes to the late symptoms [109].
Notably, the autophagic machinery cross-talks with multiple cellular processes in order to regulate ALS pathogenesis.UBQLN2 can mediate the autophagy-independent degradation of aggresomes by proteasomal machinery, while it can exacerbate NF-κB mediated inflammatory response [121].Furthermore, OPTN and p62 can regulate inflammatory response and necroptosis by interacting with TBK1 [116].

Blocked Axonal Transport
As polarized and elongated cells, motor neurons are susceptible to defects in endosomal trafficking and axonal transport due to the demand of a high turnover of membrane components, cargo-loaded vesicles, and mitochondria required for synaptic activity [122].

Cytoskeletal Proteins in ALS
At early stages, mutant SOD1 impairs the anterograde transport in a cargo-specific manner, whereby dysfunctional mitochondria dissociate from the molecular motors of axonal transport [123].Sequestration of the cytoskeletal dynein protein in stress granules (SG) by mutant SOD1 inhibits retrograde axonal transport and impairs the clearance of aggregates in neurons [124].Loss-of-function C9ORF72 mutants disrupt the actin-mediated transport of lysosomal proteins and their association with early endosomes [125].Loss-offunction OPTN mutants disrupt its interaction with myosin VI and impair the retrograde transport for autolysosome formation [126].ALS-linked mutants of the tubulinassociated protein TUBA4A perturb the dynamics and stability of microtubules [127].Mutants of profilin 1 (PFN1) disrupt its association with actin, while those in dynactin (DCTN) impair the formation of the microtubule-binding complex [109].ALS-linked mutants of the p150Glued subunit of the dynein/dynactin complex also affect the binding to microtubules [128].Notably, mutants of DCTN, PFN1, and TUBA4A are also known to dysregulate autophagy [109].While the mutant forms of SOD1 and FUS can affect kinesins by altering their expression or phosphorylation state, the ALS-associated pathologic mutants of kinesin-5A have also been reported [109,129].

Neurofilaments in ALS
Upregulation of TNF-α perturbs the kinesin activity through p38 mitogen-activated protein kinase (MAPK) signalling, and impairs the anterograde transport [130].Elevated glutamate levels impede the axonal transport of neurofilament heavy chain (NF-H) through phosphorylation [131].Importantly, the accumulation of neurofilament light and medium chain proteins (NF-L, NF-M) along the axon acts as a pathological hallmark of ALS [109].This is concomitant with the disruption of the neurofilament network and its reduced association with actin filaments and microtubules.Axonal transport defects at the presymptomatic stage impair synaptic transmission and exacerbate distal axonopathy [42].

Disruption of Endosomal Trafficking
Upon the disruption of the endosomal trafficking network, which cross-talks with autophagy and axonal transport, the viability of motor neurons (MNs) is critically impaired [122].Cytoplasmic accumulation of mutant TDP43 disrupts the receptor signalling of cytokines to induce their mis-sorting towards early or recycling endosomes, which affects synaptic growth [132].In iPSC-derived MNs from ALS patients carrying loss-of-function C9orf72 mutations, an increase has been observed in the transport of NMDA receptors and Ca 2+ -permeable AMPA receptors to the surface of dendritic spines and neurites [125].Moreover, defective transport of mannose-6-phosphate (M6P) receptors impairs the anterograde trafficking of lysosomal enzymes and cargo [125].Due to the downregulation of vacuolar protein sorting VPS26, the endosomal retromer component, aberrant endosomal recycling has been reported for the BDNF/TrkB receptor [133].These defects can arise from the disrupted interaction of C9ORF72 with Rab GTPases involved in the intracellular vesicle trafficking pathways [134].
Similar to C9ORF72, alsin (ALS2) protein is a guanine nucleotide exchange factor (GEF) for the GTPase RAB5.Loss of ALS2 impairs endosomal mobility, increases the conversion of endosomes to lysosomes, and enhances the degradation of synaptic cargo like glutamate receptors [135].Furthermore, dysregulated endosomal trafficking can be triggered by various rare mutants of VAPB, OPTN, CHMP2B, and the multivesicular body biogenesis factor ESCRTIII [7].Mutants of VCP impair endocytosis through disruption of the clathrin-associated complex, impair disassembly and autophagic degradation of SGs, and can also trigger TDP43 mislocalization [136,137].Along with autophagic dysregulation, FIG4 variants with impaired phosphatase activity can perturb the cellular abundance of phosphatidylinositol 3,5-bisphosphate, a signalling lipid involved in the retrograde transport of endosomal vesicles [138].Intriguingly, the defects in nucleo-cytoplasmic transport in ALS involve aberrant sequestration of its components in SGs, with dysfunctional RAN GTPase activity [139].FUS mutants disrupt the transportin-mediated nuclear import, whereas the nuclear egress of TDP43 and FUS mutants occurs independently of exportin-1 [140].Fig. (2) provides an overview of the dysregulated intracellular pathogenic processes across the neuron-glia network affected in ALS.

Dysregulation of RNA Metabolism and Protein Homeostasis Machinery
Motor neurons (MNs) and skeletal muscles are known to bear a metastable sub-proteome, which is characterized by the super-saturated levels of aggregation-prone proteins [141].It is majorly due to the relatively low expression of chaperones and components of the ubiquitin proteasomal system (UPS), the protein quality control machinery mechanistically integrated with autophagy [142].ALS-linked mutants of the aggregation-prone RNA-binding proteins (TDP43, FUS, SOD1) and autophagy factors (UBQLN2, OPTN, SQSTM1) aggravate their misfolding to form aggregates and fibrils [143].Notably, ALS-vulnerable neurons and muscles suffer from excessive or impaired activation of both UPS and autophagy [144,145].Cytoplasmic aggregates of ALS-linked mutant proteins act as the 'nucleation centers' for subsequent misfolding and aggregation of native proteins that results in their loss of function [143].Moreover, this generates the compact polyubiquitinated inclusion bodies that act as the immunohistopathological hallmarks of ALS cases.

Hallmark Inclusion Bodies
Ubiquitinated TDP43 aggregates are commonly present in 97% of cases of ALS and 50% of cases of ALS-FTD [146].These form hyper-phosphorylated skein-like inclusion bodies in the frontal cortex, brain stem, spinal cord, and hippocampus of TARDBP fALS or fALS-FTD and sALS cases [146].Mutant SOD1 aggregates are known to form fibrillated Lewy bodies and hyaline conglomerate inclusion bodies in SOD1 fALS and sALS cases [147].FUS aggregates produce basophilic tangle-like inclusion bodies (Bunina bodies) in the spinal cord, motor cortex, and hippocampus of FUS fALS and sALS cases [148].Interestingly, TDP43 or FUS positive inclusions are not observed in SOD1 fALS cases, although misfolding of SOD1 is triggered in both FUS fALS and TARDBP fALS cases [149].This suggests a divergent mechanism of misfolding or aggregation across various proteinopathies.Misfolded OPTN and UBQLN2 form skeinlike bodies in the frontal cortex and spinal cord in sALS cases and are also found to colocalize with TDP43 and FUS in stress granules [126,150].C9ORF72 dipeptide repeats, mainly poly(GA), form ubiquitin-positive bodies in neurons and muscles of C9orf72 fALS-FTD and sALS cases [151].This can be present with wild-type or misfolded TDP43 aggregates in the cortex and spinal cord or p62 aggregates in the hippocampus and cerebellum [151].
Overexpression or mutation of neurofilament chain proteins, dynein, peripherin, and microtubule-associated protein tau MAPT (reported in ALS-dementia-parkinsonism syndrome) have been found to induce neurofilament aggregation in ALS [7,152].Glutamate-induced overexpression of kinases (MAPK, PIN1) induces hyperphosphorylation of neurofilaments that leads to the formation of large perikaryal inclusion bodies and disruption of axonal circuitry [152].Altogether, the aggregates of misfolded proteins and neurofilaments enforce the global failure of proteasomal and autophagic degradation machinery in ALS.

Dysfunction of Ubiquitin Proteasomal System
In the early stages of ALS, a significant impairment of both total and specific catalytic activity of the ubiquitin proteasomal system (UPS) is usually observed [153].Furthermore, overexpression of the inducible proteasome subunits occurs early during disease progression, which is followed by the downregulation of the constitutive proteasome subunits [153].This process is associated with a local inflammatory response mediated by reactive gliosis [154].While the expression of 26S proteasome is unperturbed in SOD1 G93A mice, the levels of 20S proteasome components are specifically decreased in motor neurons of the lumbar spinal cord [155].Intriguingly, an upregulation of UPS activity underlies the sustained clearance of toxic protein aggregates in the skeletal muscles of SOD1 G93A mice.However, excessive activation of UPS results in muscle atrophy through the degradation of myofibrillar proteins and inhibition of myogenesis [156].

Fig. (2).
Complex interplay of molecular networks in ALS pathogenesis.Mislocalization and aggregation of misfolded proteins dysregulate RNA metabolism as well as stress granule dynamics, which in turn induces endoplasmic reticulum stress, oxidative stress, mitochondrial dysfunction, and DNA damage.Overexpression of Ca 2+ -permeable glutamate receptors in postsynaptic neurons, and increased glutamate release by presynaptic neurons and astrocytes is associated with decreased glutamate reuptake from synapse.Concomitant synaptic stripping of the inhibitory connections triggers sustained neuronal firing that results in excitotoxicity.Chemokines released from the affected neurons and reactive astroglia promote necroptosis of oligodendrocytes with subsequent demyelination.

(A higher resolution/colour version of this figure is available in the electronic copy of the article).
Aggregation-prone C9ORF72 dipeptide repeats are known to trigger neurotoxicity by impairing proteasomal activity [151].ALS-linked mutants of VCP disrupt its interaction with the proteasome and thus impair the proteolysis of bound ubiquitylated substrates [157].ALS-linked mutants of UBQLN2 induce the accumulation of UBQLN2-positive inclusions, which impair the cargo delivery to the proteasome [158].Moreover, the downregulation of the proteasomal components triggers the mislocalization and aggregation of ubiquitylated proteins and therefore has been associated with the formation of histopathological marks in ALS [143].Mutants of cyclin F (CCNF), a component of the E3ubiquitin ligase complex, induce abnormal accumulation of TDP43 in both fALS and sALS cases [159].

Dysregulation of RNA Metabolism
RNA metabolism and protein synthesis are intricately linked through the activity of dynamic stress granules, and thus there is a concomitant dysregulation of both processes in ALS [160,161].ALS-linked mutants of nuclear RNAbinding proteins TDP43, FUS, hnRNPA1, hnRNPA2B1, ATXN2, and matrin 3 (MATR3) form mislocalized cyto-plasmic aggregates because of the irreversible interactions with their prion-like low-complexity domains [162].This alters the assembly and disassembly of ribonucleoprotein granules (SG, P-bodies) that act as nucleation centers for native protein aggregation [161].
ALS-linked mutations in FUS occur mostly in the domains carrying the nuclear localization signal and intrinsically disordered regions.Similar to TDP43, such mutants mediate the cytoplasmic mislocalization of FUS [165].Aggregation of mutant FUS with nucleo-cytoplasmic shuttling proteins (HNRNPA1, HNRNPA2B1), spliceosome assembling proteins (SMN1), and mRNA processing complexes has been found to promote the dysregulation of translatome [166].Notably, C9orf72 hexanucleotide repeat expansions (HRE) induce defects in nucleo-cytoplasmic shuttling, posttranscriptional processing, ribosomal biogenesis, and assembly of stress granules [160].RNA foci with transcribed G4C2 repeats of C9orf72 can sequester other RNA-binding proteins [167].Repeat associated non-AUG (RAN) mediated translation of these transcripts and their anti-sense mRNA can produce toxic dipeptide repeats (DPRs) [168].Among these DPRs, poly(GA) mainly induces protein aggregation by impairing UPS to trigger ER stress, while poly(GR) and poly(PR) can inhibit global translation by sequestering RNAbinding proteins (FUS, TDP43, ribosomal biogenesis factors) along with their bound transcripts inside stress granules [168].
Notably, it is a widespread idea in the field that the spread of misfolding and toxic protein aggregates might underlie the course of disease progression (details in Box 3).Development of sensitive assays for the early detection of ALS-relevant misfolded proteins would allow necessary interventions for clearance before they accumulate into large degradation-resistant aggregates and thus prevent the pathological cascade of proteotoxicity.

Box 3. Cell-to-cell Propagation of ALS Pathogenicity
As per the corticofugal model, the clinical propagation of ALS is attributed to the contiguous or non-contiguous intercellular spread of toxic protein aggregates by trans-synaptic transmission [162].

The hypothesis of prion-like transmission:
Chronic cellular stress triggers prion-like conformational changes in the intrinsically disordered regions of mutant RNA/DNA-binding proteins [162].It dysregulates their liquid-liquid phase separation properties and produces irreversible aggregates that trigger the misfolding of wild-type proteins by a 'seeded aggregation' [170].The intercellular spread of 'self-perpetuating aggregates' potentially occurs through exosomal transport similar to prion transmission or through transient expression by prion-like promoters [162].
Note: Prion-like spread of mutant protein potentially accounts for the presence of TDP43 pathology in various ALS-spared regions, including motor (substantia nigra, basal ganglia) and extramotor regions (occipital lobe, amygdala, and hippocampus) [171].

Defective DNA Damage Repair System
Defective or toxic DNA damage signalling and repair (DDR), accumulation of endogenous genotoxic stressors (such as ROS), and chromatin disorganization can collectively trigger genomic instability that causes irreversible damage to the terminally differentiated motor neurons.ALS-linked mutants of various cell cycle regulator genes (NEK1, C21ORF2, CCNF) are known to induce abnormalities in DNA damage repair [172].Mutants of VCP impair DNA damage response through the loss of interaction with nonhomologous end-joining repair (NHEJ) factors Ku70/80 and ring finger protein 168 (RNF168) [172].These defects can be exacerbated by mutants of SQSTM1 through the loss of interaction with other NHEJ factors, such as ataxia telangiectasia mutated (ATM) and RAD50 [172,173].In ALS/ FTD cases, cytoplasmic mislocalization of TDP43 impairs the nuclear transport of the DNA repair complex of X-ray repair cross-complementing complex 4 (XRCC4) and DNA ligase IV (LIG4), which results in toxic accumulation of DNA double-strand breaks (DSBs) [174].Additionally, mutant FUS impairs the recruitment of the DNA repair complex XRCC1/LIG3 to DNA single-strand breaks (SSBs) following oxidative damage, which impairs the base excision repair (BER) [175].
Intriguingly, mutant TDP43 and FUS dysregulate DNA: RNA hybrid (R-loop) formation and thus promote transcriptional stress [176].Transcription of C9orf72 repeat expansions facilitates the accumulation of R-loops as well as the formation of toxic DNA secondary structures, hairpins, and G-quadruplexes [177].C9orf72 RNA repeat expansion (RRE) foci sequester various RNA-binding proteins (TDP43, FUS, NPM1, APE1), and thus impairs their DNA damage repair activity [178].Moreover, dipeptide repeats secreted by C9orf72 fALS astrocytes can trigger genomic instability by sequestering DNA repair factors, inducing R-loop formation, and chromatin compaction [179].ALS-linked mutants of the R-loop processing factors such as SETX, ATXN2, and HNRNPD are well known [180].Furthermore, neuronal accumulation of C9ORF72 dipeptide repeats at the nuclear membrane can trigger chromatin disorganization [181].In both fALS and FTD cases, TBK1 haploinsufficiency leads to chromosomal instability.Cytoplasmic accumulation of ssD-NA in ALS-affected neurons over-activates the cyclic GMP/ AMP synthase (cGAS) and stimulator of interferon gene (STING) signaling to trigger a proinflammatory response [182].Mutants of other ALS-relevant proteins (MATR3, ERBB4), as well as disease modifiers like histone acetyltransferase ELP3, mRNA export factor GLE1, and stress granule component TIA1, have also been found to deregulate DNA damage repair [172].

Dyshomeostasis of Endoplasmic Reticulum-Mitochondria Functional Networks
Due to the continuous firing of ALS-vulnerable motor neurons (MNs), the chronic depletion of Ca 2+ reserves in the endoplasmic reticulum (ER) leads to ER stress [183].Concurrently, the chronic Ca 2+ overload in mitochondria activates pro-apoptotic factors to trigger the release of cell death signals [183].Loss-of-function mutations in TARDBP and FUS are known to impair autophagosome formation by inter-fering with ER-Golgi transport and ER-mitochondria interactions.These proteins also compromise mitophagy through the sequestration of the E3 ubiquitin ligase PARKIN [184].Notably, the dysfunction of selective autophagy contributes to RNA metabolism dysregulation, ER stress, and mitochondrial damage in motor neurons [185].Accumulation of ROS further inhibits autophagy, stabilises misfolded protein aggregates, and promotes inflammation.This entire loop exacerbates ER stress and mitochondrial damage that results in apoptosis [186].

ER-associated Dysfunction
Astrocytic deletion of membralin, a factor involved in ER-associated degradation (ERAD), facilitates neuronal excitotoxicity [84].Mutant SOD1 is known to dysregulate the stress granule formation by colocalizing with its core components like RasGTPase-activating protein-binding protein 1 (G3BP1) and T-cell intracellular antigen 1 (TIA1) [187].At the early symptomatic stage, a selective induction of the PRKR-like ER kinase (PERK) arm of neuronal unfolded protein response (UPR) has been reported [188].However, this fails to prevent misfolding of mutant proteins due to the naturally low levels of ER-resident chaperones.In transgenic SOD1 mice and sALS patients, the UPR machinery upregulates protein disulphide isomerase (PDI) and other ERresident chaperones (protein disulphide isomerase PDIA3) in astrocytes and oligodendrocytes [189].Furthermore, inactive proteins sequestered in stress granules suppress the general translation process and ERAD pathways [190].Aggregation of mutant VAPB, an integral ER membrane protein, dysregulates the activating transcription factor 6 (ATF6) and X-box binding protein 1 (XBP1) arms of UPR [191].Despite being an initial adaptive response to ER stress, the prolonged activation of UPR leads to apoptosis [188].

Golgi-associated Dysfunction
ALS-associated mutant proteins can induce Golgi fragmentation by the inhibition of vesicular trafficking in the ER-Golgi and Golgi-plasma membrane networks [192].Given that the axonal Golgi networks regulate the local synthesis and trafficking of axonal membrane proteins, the Golgi fragmentation can disrupt axonal secretory transport and autophagy [192].Notably, the fragmentation of somatic and dendritic Golgi precedes axonal retraction and muscle denervation in ALS mouse models [193].

Mitochondrial Dysfunction
In ALS patients, Ca 2+ homeostasis is disrupted in the distal dendrites and axons due to the block of mitochondrial transport, with concurrent mitochondrial accumulation in the soma and proximal axon hillock [194].MNs derived from the spinal cord of ALS patients and presymptomatic SOD1 G93A mice show mutant SOD1 aggregates in intermembrane space, mitochondrial swelling, fragmentation of the mitochondrial network, peroxidation of mitochondrial membrane lipids, decreased coupling between mitochondrial oxidative phosphorylation and electron transport chain (ETC), and reduced expression of mitochondrial enzymes [195].Downregulation of peroxisome proliferatoractivated receptor gamma coactivator 1α (PGC-1α), a master regulator of mitochondrial biogenesis and function, exac-erbates disease progression across CNS and skeletal muscles of ALS [196].At the disease onset, dense clusters of presynaptic mitochondria are observed in spinal neurons due to the downregulation of mitochondrial transporter MIRO1 [197].Moreover, TDP43 inclusion bodies interact with critical mediators of mitochondrial dynamics and mitophagy, such as prohibitin 2 (PHB2) [198].ALS-linked mutants of VAPB and VCP disrupt the mitochondrial-ER contacts (MERCs) to trigger organellar dysfunction [199].Dysfunction of mitochondrial complex I and complex V subunits of ETC has been observed in C9orf72 ALS [200].

Oxidative Stress
In the astrocytes derived from ALS patients and mouse models, there is a decrease in the nuclear factor erythroid 2related factor 2 (Nrf2)-dependent antioxidant signalling.This is associated with the depletion of glutathione, upregulation of peroxiredoxins and catalase activity, and persistent microglial inflammatory damage to the neighbouring neurons [201].In the motor cortex, depletion of mitochondrial glutathione reductase (GSH) potentially arises from sustained glutamatergic signalling, and in turn, facilitates mitochondrial Ca 2+ release.This leads to dysfunction of the voltagedependent anion channel (VDAC) and adenine nucleotide translocator (ANT), along with the opening of apoptotic mitochondrial permeability transition pore (mtPTP) channel in ALS-affected neurons [202].Mitochondrial ROS accumulation induces glial glutamate release by the upregulation of cysteine-glutamate antiporters, which further promotes oxidative stress due to decreased cysteine uptake [203].Furthermore, the presynaptic decline at NMJs is facilitated by ROS released by the glial cells and infiltrated immune cells [204].Cytoplasmic mislocalization of mutant SOD1 impedes the transcription of genes involved in DNA damage response and antioxidant pathways [205].Mutant TDP43 induces oxidative stress through the nuclear accumulation of Nrf2 and activation of mitochondrial UPR machinery.In turn, oxidative stress promotes GADD34-mediated phosphorylation as well as aggregation of acetylated TDP43 [206].Mutant FUS inhibits the repair of oxidative stress-mediated DNA damage through defects in DNA ligation [201].Similar to the ALSlinked mutants of D-amino acid oxidase (DAO) and ALS2 proteins, the toxic dipeptide repeats of C9ORF72 promote ROS generation [172].Understandably, the exogenous prooxidative stress factors such as intense physical activity, radiation, metal toxicity etc., as well as endogenous modifiers such as malnutrition, cachexia, respiratory weakness etc., can exacerbate the disease progression in ALS [207].

Dysregulation of Metabolic Programming
Rapid cycles of action potential propagation and presynaptic transport across the long axon of fast-firing motor neurons are major factors for the high energy demand of CNS [37,208].Moreover, high ATP levels are required to prevent the aggregate formation in both soma and axon [209].Neuronal energy homeostasis is linked with the antioxidant glutathione machinery through a fine balance between the glycolytic and pentose phosphate pathway (PPP) flux [210].High metabolic demand and low oxidative stress response of corticospinal neurons and skeletal muscles render them se-lectively vulnerable to the energy defects in ALS.This impairs self-regenerative pathways, which facilitates terminal nerve retraction and loss of reinnervation [211].In order to produce in situ energy substrates during impaired neuronal glucose uptake, ROS generated by the mitochondrial ETC system stimulates lipid transport to astrocytes, which otherwise accelerates neurodegeneration [212].
In ALS patients with pure UMN involvement, positron emission tomography (PET) imaging using 2-deoxy-2fluoro-D-glucose ( 18 FDG) revealed glucose hypometabolism across the fronto-dorsolateral cortex, precentral cortex, and temporal cortex [213].However, a diverse glucose hyper-metabolism pattern is found in the midbrain, spinal cord, and skeletal muscles of ALS patients [214,215].Severe metabolic impairment occurs in the precentral gyrus during the bulbar onset and in the cervical cord during the upper limb onset [214].Therefore, diverse states of metabolic pathogenicity occur spatiotemporally across CNS during the disease progression.In SOD1 G93A mice, glucose uptake in the spinal cord increases at the presymptomatic stage, but it declines progressively [216].In the spinal cord of endstage ALS patients and SOD1 G93A mice, the uncoupling of blood flow and glucose metabolism is observed with a concomitant increase in glycogen storage [217].There is a persistent downregulation of glycolysis rate-limiting enzyme phosphofructokinase 1 (PFK1), Kreb's cycle enzyme succinate dehydrogenase (SDH), and Kreb's cycle intermediates in the CNS and skeletal muscles [218,219].Interestingly, impaired neuronal glucose oxidation has been associated with unperturbed astrocytic Kreb's cycle at symptomatic stages in SOD1 G93A mice [220].Dysregulation of nucleotide metabolism and histidine metabolism during ALS progression might serve as a metabolic signature of pervasive DNA damage by oxidative stress [180].

'Hyper' Metabolism in ALS
Systemic hypermetabolism, accompanied by dyslipidaemia, has been reported at the presymptomatic stage in ALS patients and SOD1 G93A mice [221].The shift from glucosedependent to lipid-based metabolism may be an adaptive response against the disrupted energy homeostasis at early stages [222].Ketosis in astroglia promotes the reuptake of extracellular glutamate and induces the synthesis of GABA from glutamine in neurons [223].Notably, endogenous energy reserves of skeletal muscles and adipose tissues are progressively depleted during the course of the disease.As a result, ALS patients often develop insulin resistance due to the dysregulated glycogen metabolism in muscles [224].Proteomic analysis of motor neurons from SOD G93A mice revealed the upregulation of hydroxy acyl-CoA dehydrogenase subunit A (HADHA) and acetyl-CoA acetyltransferase 2 (ACAT2) enzymes involved in ketosis, and βhydroxy β-methyl glutaryl-CoA (HMG-CoA synthase) involved in cholesterol metabolism [225].Interestingly, lipid accumulation during ALS progression is independent of the downregulation of enzymatic and transcriptional regulators of lipid synthesis [226].
In the spinal cord of transgenic SOD1 G86R mouse, inhibition of β-glucocerebrosidase is found to promote glycosphingolipid metabolism, which delays ALS onset by inducing muscle reinnervation [227].Interestingly, the ganglioside GM1a interacts with the BDNF receptor TrkA and with the Na + /Ca 2+ exchanger at the nuclear membrane to facilitate Ca 2+ transfer to ER [228].Towards later stages, prolonged lipid hypermetabolism promotes oxidative stress via the byproducts of ketosis and cholesterol synthesis in the spinal cord and skeletal muscles [226].Notably, the progressive decrease of membrane fluidity observed in ALS mouse models can be attributed to the increased peroxidation of polyunsaturated fatty acids, which generates excitotoxic intermediates like 4-hydroxynonenal, and inflammatory mediators like eicosanoids [229].Moreover, the excessive production of cholesterol and sphingolipids impairs vesicular transport, neurotransmitter cycling, and cytoskeletal organization and triggers excitotoxicity through defective RNA editing of GLUR2 transcript in neurons [230].This process disrupts the 'cholesterol sensor' LXR signalling, which induces apoptosis [231].
Multiple pilot clinical trials have suggested that a highfat diet can improve prognosis and delay disease progression in ALS patients by promoting LDL/HDL ratios or body mass index [232].Importantly, approaches that target various steps of the neuroglial glucose metabolism might have potential in ALS therapy [218].Fig. (3) provides an overview of the noncell autonomous and cross-talking pathogenic processes across the neuroglial networks and neuromuscular junctions affected in ALS.

GENE & ENVIRONMENT: EPIGENETIC & EPI-TRANSCRIPTOMIC LANDSCAPE IN ALS
Only ~30 environmental risk factors have been significantly associated with ALS incidence till date through summary statistics from genome-wide association studies, linkage disequilibrium score regression, and Mendelian randomization analyses [16].These factors include intense physical activity, psychiatric disorders, military service, contact sports, comorbid diseases (Type I diabetes, viral infections), and exposure to neurotoxins, heavy metals and organic solvents [233].As per the gene-environment-time interaction (GETI) hypothesis of ALS incidence, a genetic risk variant can interact with a combination of environmental risk factors at a specific age in a particular geographical region for a definite period of time [16].A panel of inherited genetic defects with varying penetrance might differentially trigger the primary pathogenic pathway.It needs to be complemented by variable intensities and combinations of environmental triggers to affect the secondary pathways that cumulatively set off the pathological cascade for ALS onset [234].
Epigenetic modifications involve the reversible heritable or non-heritable alterations in gene expression without any changes to the genome sequence, in response to endogenous and environmental factors [235].Changes in the epitranscriptome (RNA epigenetics) reveal a novel code of locus and cell type-specific modifications on different classes of RNA for a dynamic and rapid co-transcriptional and posttranscriptional regulation of RNA expression, stability, localization, and function [236].

Epigenetic Signatures in ALS
In spinal LMNs of sALS and C9orf72 fALS patients, immunohistochemical studies revealed an increase in methylation and hydroxymethylation of CpG islands at gene promoters, which inversely correlated with TDP43 pathology [237].Quantitative analyses of methylation profiles depicted that 5'-hydroxymethylcytosine (5hmC) levels are increased in corticospinal motor neurons of hSOD1 G93A mice and decreased in prpTDP43 A315T mice [238].Repeat-primed and methylation-specific PCR revealed hypermethylation of CpG islands near the C9orf72 repeat expansions in blood and brain samples from 10-30% of ALS and FTD cases [239].Notably, increased 5'-methylcytosine (5mC) levels in mitochondria (especially at 16S rRNA gene) and downregulation of DNA methyltransferase DNMT3A, associated with mitophagy, were reported in skeletal muscles and spinal motor neurons of transgenic SOD1 mice [240].
In ALS patients, deficiency of the histone acetyltransferase (HAT) elongator acetyltransferase complex subunit 3 (ELP3) leads to a decrease in the transcriptional activation acetylation marks on lysine 14 of histone H3 (H3K14ac) and lysine 8 of histone H4 (H4K8ac) [241].In SOD1 G93A mice, inhibition of the histone deacetylase HDAC6 at early stages increases α-tubulin and TDP43 acetylation, while HDAC6 overexpression at late stages promotes autophagy [242,243].In skeletal muscles of SOD1 G93A mice and ALS patients with rapid disease progression, HDAC4 is upregulated at presymptomatic stages and protects from skeletal muscle atro-phy but is decreased upon symptom onset [244].In primary cultures of spinal motor neurons of transgenic FUS R495X mice, downregulation of protein arginine N-methyl transferase 1 (PRMT1) is associated with a decrease in asymmetric di-methylation on arginine 3 of histone H4 (H4R3me2asym), with concomitant downregulation of H3K9ac and H3K14ac marks [245].ALS-linked TDP43 M337V and FUS R495X mutants have been found to reduce the global levels of conjugate H3 phosphorylation at serine 10 and acetylation at lysine 14 (H3S10Ph-K14Ac), which in turn dysregulates NF-κB signalling [246].Distinct alteration patterns of the cross-talking histone methylation and histone acetylation marks suggest that each proteinopathy dictates its distinct histone modification landscape to converge on the pathological hallmarks of ALS.Despite a wide variety of studies regarding miRNA deregulation in ALS patients, there is a limited overlap of the miRNA signatures, possibly because of the variation in study populations, types of control subjects, disease stages, sample sources, methods of tissue extraction, and miRNA expression profiling [247].A recent meta-analysis of all available studies on miRNA dysregulation in ALS has identified four miRNAs (hsa-miR-9-5p/3p, hsa-miR-28-5p/3p, hsa-miR-132-5p/3p, hsa-miR-146a-5p/3p) to be the most commonly reported across nervous tissue, cerebrospinal fluid, skeletal muscles, and plasma or blood or serum [248].A competitive endogenous circRNA-miRNA-mRNA axis reported in ALS involves the circRNA hsa-circ-0023919 that inhibits miR-9, which otherwise regulates the transcription of intermediate filament NEFL and leads to neurofilament aggregation [249].

Epitranscriptomic Landscape of ALS
Molecular heterogeneity in ALS etiopathogenesis can potentially be attributed to aberrant RNA modifications on various RNA substrates or a modification-independent function of the RNA modifiers [250].In the spinal cord of ALS patients and transgenic SOD1 mice, accumulation of oxidized mRNA species bearing 8-oxo-7,8-dihydroguanosine (8OHG) has been consistently observed [251].Oxidation of mRNA species has been primarily found in motor neurons and oligodendrocytes at the early presymptomatic stage, before the oxidation of lipids, proteins, and DNA during the symptomatic stage [251].Post-transcriptional adenine-toinosine (A-to-I) RNA editing at the glutamine/arginine (Q/R) site in GluA2, a subunit of glutamate receptor GluR2, is essential to prevent excitotoxicity in spinal motor neurons of sALS patients [252].Upon conditional knockout of the RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) in mice, the motor neurons expressing Q/R siteunedited GluA2 subunit undergo apoptosis due to calcium overload and dysregulated autophagy [253].The mislocalization of ADAR2 has been reported in the motor cortex and lumbar spinal cord of C9orf72 ALS/FTD patients [254].
Through the technique of methylated RNA immunoprecipitation with sequencing (MeRIP-seq), a global decrease in the level of N6-methyladenosine (m 6 A) modification on RNA has been observed upon TARDBP knockdown in mammalian cells [255].This phenomenon is similar to that observed upon silencing of the m 6 A writer METTL3, which regulates synaptic growth at neuromuscular junctions [255].Interestingly, mutations in the m 6 A reader hnRNPA2B1, which also regulates proteostasis, have been frequently associated with ALS [256].Whole-genome sequencing revealed a positive association of motor neuron-specific variants in fat mass and obesity-associated protein (FTO), an m 6 A eraser factor from the α-ketoglutarate dependent dioxygenase family, with sALS cases [257].Upon the loss of site-specific m 5 C modifications during cellular stress, the ALS-associated ribonuclease ANG promotes the cleavage of tRNAs to generate tRNA-derived small RNAs (tsRNAs) and triggers the assembly of stress granules [258].Deregulation of the RNA methyltransferase DNMT2, which otherwise prevents tsRNA accumulation has been observed in FUS fALS cases with a concomitant increase in 5mC mark on the proximal FUS promoter, a lower FUS expression and a higher cytoplasmic FUS aggregation in motor neurons [259].Notably, accumulation of 5'ValCAC tRNA fragment (valine tRNA with CAC anticodon) with increased ANG expression has been reported in the spinal cord and serum from SOD1 G93A mice at the symptom onset [260].A recently discovered hallmark of ALS-FTD cases involves the nuclear depletion and cytoplasmic accumulation of the neuronal-enriched splicing factor SFPQ, which produces aberrant intron-retaining (IR) transcripts of neuronal proteins for accumulation in RNA granules at neurites [261].A broad overview of commonly reported epigenetic and epitranscriptomic signatures of ALS pathogenesis is given in Fig. (4).

CHALLENGES AND PROSPECTS FOR ALS PATHOMECHANISTIC RESEARCH
The Food and Drug Administration (FDA) has currently approved very few drugs, namely Riluzole, Edaravone, and NueDexta, that can only moderately improve survival across specific ALS patient populations.Widespread efforts for treatments targeting the pathomechanistic networks of ALS have led to several late-stage clinical trials, with the primary therapeutic target being neuroinflammatory pathways (particularly the complement system) followed by oxidative and proteotoxic stress response [262].These involve inhibitor molecules, genetic therapies through antisense oligonucleotides (ASO), stem-cell-based therapies, and recombinant humanized monoclonal antibodies [263].Notably, the tyrosine kinase inhibitor Masitinib has been reported to prolong survival in phase III clinical trials by targeting macrophages, mast cells, and microglia [264].For a detailed review of previously tested and failed treatment strategies, and novel therapeutics (including the repurposing of available drugs) in the pipeline, the readers can refer to Corcia et al. 2021 [263] and Mead et al. 2023 [262].

Broader Relevance of Pre-clinical Models
The corticospinal tract in rodents runs through the dorsal funiculi and ends at the dorsal horn, as opposed to the direct ventral connections in humans [265].ALS models of nonprimates show early corticomotor hyperexcitability, which modulates LMN dysfunction through the local oligosynaptic and excitatory inter-neuronal networks [266].It is important to use a range of complex disease models, including patient fibroblast-derived neural progenitor differentiation systems, and monitor the same prognostic phenotype marker from disease models to clinical trials.

Stage-specific Non-cell Autonomous Phenotypes
In-depth methodological analysis and holistic integration of epidemiological and clinicopathological data from patient cohorts can help to characterize common and divergent mechanisms in ALS pathophysiology.Modulation of the activity of γ-MNs, Renshaw cells, or metabotropic GluRs with the help of inhibitory 'designer receptors exclusively activated by designer drugs' (DREADDs) might be useful in targeting the neuronal microcircuit dysfunction in ALS [61].Notably, the lack of selectivity and efficacy of the autophagy-inducer drugs tested for ALS therapy makes it necessary to elucidate the site and the stage-specific window of dysfunction [109].Disaggregases like heat shock proteins HSP27 and HSPB1 can regulate phase separation and liquidto-gel transition of accumulated TDP43 or stress granule protein G3BP1 condensates in axons and NMJs [267].The use of 3D microfluidic system models for the stratification of heterogeneous non-neuronal phenotypes may provide new therapeutic targets to combat the non-cell autonomous pathogenicity in ALS [71].

Fig. (4).
Changes in the epigenetic and epitranscriptomic landscape during ALS pathogenesis.ALS-associated epigenetic signatures include a global increase in DNA methylation with a concomitant deregulation of DNA methyltransferases (DNMTs).A global decrease in histone acetylation is concomitant with a dysregulation of histone acetyltransferases and histone deacetylases (HDACs).Global dysregulation of miRNA levels includes their sequestration in stress granules by mutant protein aggregates.ALS-relevant epitranscriptomic modifications commonly involves A-to-I editing defect in GluA2 transcript, which upregulates Ca 2+ -permeable glutamate receptor in neurons.Accumulation of ROS-induced oxidized mRNA species, and tRNA fragment cleaved by mutant ANG impairs protein translation efficiency.Aberrant intron-retaining transcripts promote mislocalization of RNA binding proteins.Abbreviations: CHD2: chromodomain DNA helicase protein 2; CBP/p300: CREB binding protein p300; EAAT2: excitatory amino acid transporter 2; nBAF: neuronal Brg1-associated factor; PRMT1: protein arginine methyltransferase 1; SIRT: sirtuin; SFPQ: splice factor proline glutamine rich.(A higher resolution/colour version of this figure is available in the electronic copy of the article).
It is sensible to design novel therapies that either target the inhibition of degenerative factors selectively active in ALS-vulnerable motor neurons and myofibres or the activation of intrinsic neuroprotective defence factors expressed in their resilient counterparts [52].In the early stages of ALS, the synaptic hyperexcitability overactivated BDNF/TrkB signaling, which can be prevented by the inhibition of the adenosine 2A receptor (A 2a R) that transactivates TrkB [268].Development of high-resolution techniques that account for spatial subdivision of metabolic identities across heterogeneous cellular networks is required for the 'precision medicine' research in ALS.Machine learning techniques can prove useful in the identification of biomarkers of target engagement as well as disease progression [269].

Stratification of Sporadic Cases
There is a need for integration of the effect of genome, exposome, and behavome on the spatiotemporal patterns of health outcomes across populations.This brings in the concept of genetic geographic information science (Genetic GISc) [270].Variations in the neuroendocrine stress response can influence the clinical heterogeneity of ALS in a vulnerable genetic, constitutional, or epigenetic background [271].

Role of Hormetic Dose Response in Pathology
An interesting hypothesis of ALS pathogenecity posits that the accumulation of age-associated oxidative damage accounts for the late onset and slow progression of neurodegenerative disorders [272].Mild exposure to environmental stressors activates the adaptive stress response pathways involving the vitagene network, through a central redoxsensing thiol-modifying signalling, for the synthesis of stress-resistance proteins [273].This integrated response protects the biological system from severe exposure in the future and constitutes the biphasic 'preconditioning' or 'hormetic' response [274].Intriguingly, improvement of prognosis in SOD1 G93A mice observed upon exposure to a sub-toxic dose of neurotoxin -methylamino-L-alanine (L-BMAA) suggests that variation in exposure to an environmental risk factor can be neuroprotective or neurotoxic in the context of a genetic predisposition [275].Sustained activation of NMDAR leads to prolonged stimulation of NOS, generation of superoxide or peroxynitrite anions, nitric oxide (NO) mediated S-nitrosylation of survival proteins (e.g., MMP9, PDI, COX) and glutamate release-associated synaptic vesicle proteins [272].However, physiological levels of NO promote survival by nitrosylating NMDAR and Caspase-3 subunits with activation of Akt kinase and CREB signalling [272].The adaptive nature of hormetic dose responses provides a quantitative index of the plasticity of biological systems irrespective of gender, biological model, timepoint, nature of the agent, or type of induction [274].To combat both acute and chronic neurodegenerative diseases, the lowdose response stimulation of ER stress and antioxidant pathways can be targeted through dietary restriction or chemically modified drugs [273].Nutraceuticals such as hydroxytyrosol (Hidrox ® ) can be promising in ALS therapy since it modulates the Nrf2-regulated hemeoxygenase-1 (HO-1) pathway, NF-κB signalling, and sirtuin signalling [276].Moreover, resveratrol, an activator of SIRT1-FOXO signalling and Keap1-Nrf2 signalling, is known to be neuroprotective in experimental ALS models [277].The range estimation of stimulatory hormetic dose responses can be used to improve the quantitative design of a clinical study, which increases the probability of inducing an optimal response across the heterogenous patient subgroups [278].

Epigenetic and Epitranscriptomic Precision Research
Novel techniques to detect disease-associated epigenetic marks include methylcytosine-capture DNA hybridization immunoassay for semi-quantitative detection of the repeat methylation levels, especially for the diagnosis of C9orf72 repeat expansion carriers [279].Notably, a recent study demonstrated direct in vivo non-invasive spatial profiling of DNA methylation in mouse brains through a novel 13 C tracer-based magnetic resonance spectroscopic imaging ( 13 C-MRSI) method [280].Regarding ALS epigenetic therapy, research should now be directed toward drugs targeting DNA and histone methylation modifiers and histone phosphorylases.Inhibition of protein phosphatase 1, which erases the H3S10ph mark, can be studied as a potential therapeutic target [281].Recently, FDA has approved AMX0035 (Relyvrio or Albrizo) for ALS treatment, a combination of taurursodiol and the pan-HDAC inhibitor sodium phenylbutyrate (4-PB), which rescues from ER stress and mitochondrial dysfunction [282].However, large-scale clinical trials for epigenetic modifier drugs is limited, given the opposing nature of epigenetic modifier activity during ALS and the risk of off-target effects [283].
A bidirectional dCasRx epitranscriptome editing platform composed of a nuclear-localized dCasRx conjugated with methyltransferase or demethylase is currently used to discern the molecular function of individual site-specific m 6 A modifications or modifiers [284].RNA metabolism defects have been successfully reversed by CRISPR-Cas9 targeting of repeat expansions in C9orf72 fALS patient cells [285].Given the cross-talk between epigenetic and epitranscriptomic signatures, it would be prudent to design 'precision medicine' regimens for sALS patient subgroups with a defined genetic makeup and exposome.

Questions for Future Research
• How do the shared and unique molecular networks of pathogenicity determine a longer survival in ALS cases involving the male gender, spinal form of onset, younger age at onset or diagnosis, and a higher baseline score on the revised ALS Functional Rating Scale (ALSFRS-R) or the Rasch-built Overall ALS Disability Scale (ROADS)?
• How can different environmental factors influence common metabolic pathways to induce changes in the epigenetic or epitranscriptomic landscape in a tissue or cell-specific manner?
Rather than simple association studies, interactions between cellular processes and molecular networks need to be considered for combination therapy or targeting upstream regulators in complex neurodegenerative disorders like ALS.

CONCLUSION
ALS entails a complex genetic architecture that is oligogenic or polygenic in sporadic cases and mostly monogenic in familial cases.Secondary oligogenic variants with low allele frequency have uncertain or varying effect sizes in the presence of a highly penetrant primary variant, which might result in the pleiotropy of phenotype [286].Based on various large-scale epidemiological studies, the ALS phenotype has been regarded as the culmination of a six-step process of pathogenesis, with a lesser number of pathogenesis-relevant steps in patient cohorts with large-effect genetic variants (SOD1, C9ORF72) [287].The disease endophenotype is the end result of the interaction between inherited genetic risk factors and a combination of environmental and endogenous risk factors.The genetic architecture of ALS can be partitioned into distinct risk-associated components rather than simply fALS or sALS, which in turn can facilitate the validation of pathological mechanisms and the stratification of patient sub-groups.The combinatorial effect of interactions between various ALS-linked genetic variants and environmental risk factors can regulate pathogenesis in a synergistic or antagonistic manner [16,287].

AUTHORS' CONTRIBUTIONS
Bedaballi Dey conceived the idea for the article, performed the literature survey, and wrote the first draft of the manuscript.Anant Bahadur Patel conceived the idea for the article, critically revised and made final editing to the work.Arvind Kumar conceived the idea for the article, critically revised and made final editing to the work.All authors read and approved the final manuscript.

Fig. ( 3
Fig. (3).A multi-network and non-cell autonomous failure of cellular homeostasis processes drives ALS.Misfolded protein aggregates induce defects in the intracellular homeostasis processes, which in turn disrupts the protective neuron-astroglia crosstalk.Concomitantly, the defects in synaptic scaling and plasticity of motor neurons results in glutamate excitotoxicity.Release of neuron-specific antigens and proinflammatory stimuli primes T-helper cells into a proinflammatory Th17 phenotype for recruitment into the central nervous system.Side by side, infiltration of cytotoxic T-cells and natural killer cells occurs through adaptive immune signalling.Defective axonal transport hinders the cholinergic transmission towards neuromuscular junctions and impairs myofibre contractility.Release of growth-cone repellents from skeletal muscles and chemorepellents from dying terminal Schwann cells triggers distal axonopathy.(A higher resolution/colour version of this figure is available in the electronic copy of the article).